RESUMO
Background: Andrographolide (Andro), an extract of Andrographis paniculate (Burm.f.) Wall. ex Nees (Acanthaceae), possesses diverse biologically active properties. However, the precise mechanisms and effects of Andro on pancreatic cancer (PC) remain unclear. Methods: The cytotoxic potential of Andro and underlying mechanism towards PC cells was investigated through in vitro experiments and a xenograft mouse model. PC cells were first subjected to varying concentrations of Andro. The reactive oxygen species (ROS) was assessed using flow cytometry and DCFH-DA staining. The apoptosis rate was detected by flow cytometry. Additionally, western blot was applied to evaluate the expression levels of cleaved-caspase-3, DJ-1, LC3-I, LC3-II, and p62. To further elucidate the involvement of ROS accumulation and autophagy, we employed N-acetylcysteine as a scavenger of ROS and 3-Methyladenine as an inhibitor of autophagy. Results: Andro demonstrated potent anti-proliferative effects on PC cells and induced apoptosis, both in vitro and in vivo. The cytotoxicity of Andro on PC cells was counteracted by DJ-1 overexpression. The reduction in DJ-1 expression caused by Andro led to ROS accumulation, subsequently inhibiting the growth of PC cells. Furthermore, Andro stimulated cytoprotective autophagy, thus weakening the antitumor effect. Pharmacological blockade of autophagy further enhanced the antitumor efficacy of Andro. Conclusion: Our study indicated that ROS accumulation induced by the DJ-1 reduction played a key role in Andro-mediated PC cell inhibition. Furthermore, the protective autophagy induced by the Andro in PC cells is a mechanism that needs to be addressed in future studies.
Assuntos
Apoptose , Autofagia , Diterpenos , Neoplasias Pancreáticas , Proteína Desglicase DJ-1 , Espécies Reativas de Oxigênio , Espécies Reativas de Oxigênio/metabolismo , Diterpenos/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Autofagia/efeitos dos fármacos , Proteína Desglicase DJ-1/metabolismo , Proteína Desglicase DJ-1/genética , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos NusRESUMO
PARK7, also known as DJ-1, plays a critical role in protecting cells by functioning as a sensitive oxidation sensor and modulator of antioxidants. DJ-1 acts to maintain mitochondrial function and regulate transcription in response to different stressors. In this study, we showed that cell lines vary based on their antioxidation potential under basal conditions. The transcriptome of HEK293 cells was tested following knockdown (KD) of DJ-1 using siRNAs, which reduced the DJ-1 transcripts to only 12% of the original level. We compared the expression levels of 14k protein-coding transcripts and 4.2k non-coding RNAs relative to cells treated with non-specific siRNAs. Among the coding genes, approximately 200 upregulated differentially expressed genes (DEGs) signified a coordinated antiviral innate immune response. Most genes were associated with the regulation of type 1 interferons (IFN) and the induction of inflammatory cytokines. About a quarter of these genes were also induced in cells treated with non-specific siRNAs that were used as a negative control. Beyond the antiviral-like response, 114 genes were specific to the KD of DJ-1 with enrichment in RNA metabolism and mitochondrial functions. A smaller set of downregulated genes (58 genes) was associated with dysregulation in membrane structure, cell viability, and mitophagy. We propose that the KD DJ-1 perturbation diminishes the protective potency against oxidative stress. Thus, it renders the cells labile and responsive to the dsRNA signal by activating a large number of genes, many of which drive apoptosis, cell death, and inflammatory signatures. The KD of DJ-1 highlights its potency in regulating genes associated with antiviral responses, RNA metabolism, and mitochondrial functions, apparently through alteration in STAT activity and downstream signaling. Given that DJ-1 also acts as an oncogene in metastatic cancers, targeting DJ-1 could be a promising therapeutic strategy where manipulation of the DJ-1 level may reduce cancer cell viability and enhance the efficacy of cancer treatments.
Assuntos
Técnicas de Silenciamento de Genes , Imunidade Inata , Proteína Desglicase DJ-1 , Humanos , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/metabolismo , Imunidade Inata/genética , Células HEK293 , Mitocôndrias/metabolismo , Mitocôndrias/genética , RNA Interferente Pequeno/genética , Transcriptoma , Regulação da Expressão Gênica , Perfilação da Expressão GênicaRESUMO
Variants in seven genes (LRRK2, GBA1, PRKN, SNCA, PINK1, PARK7 and VPS35) have been formally adjudicated as causal contributors to Parkinson's disease; however, individuals with Parkinson's disease are often unaware of their genetic status since clinical testing is infrequently offered. As a result, genetic information is not incorporated into clinical care, and variant-targeted precision medicine trials struggle to enrol people with Parkinson's disease. Understanding the yield of genetic testing using an established gene panel in a large, geographically diverse North American population would help patients, clinicians, clinical researchers, laboratories and insurers better understand the importance of genetics in approaching Parkinson's disease. PD GENEration is an ongoing multi-centre, observational study (NCT04057794, NCT04994015) offering genetic testing with results disclosure and genetic counselling to those in the US (including Puerto Rico), Canada and the Dominican Republic, through local clinical sites or remotely through self-enrolment. DNA samples are analysed by next-generation sequencing including deletion/duplication analysis (Fulgent Genetics) with targeted testing of seven major Parkinson's disease-related genes. Variants classified as pathogenic/likely pathogenic/risk variants are disclosed to all tested participants by either neurologists or genetic counsellors. Demographic and clinical features are collected at baseline visits. Between September 2019 and June 2023, the study enrolled 10 510 participants across >85 centres, with 8301 having received results. Participants were: 59% male; 86% White, 2% Asian, 4% Black/African American, 9% Hispanic/Latino; mean age 67.4 ± 10.8 years. Reportable genetic variants were observed in 13% of all participants, including 18% of participants with one or more 'high risk factors' for a genetic aetiology: early onset (<50 years), high-risk ancestry (Ashkenazi Jewish/Basque/North African Berber), an affected first-degree relative; and, importantly, in 9.1% of people with none of these risk factors. Reportable variants in GBA1 were identified in 7.7% of all participants; 2.4% in LRRK2; 2.1% in PRKN; 0.1% in SNCA; and 0.2% in PINK1, PARK7 or VPS35 combined. Variants in more than one of the seven genes were identified in 0.4% of participants. Approximately 13% of study participants had a reportable genetic variant, with a 9% yield in people with no high-risk factors. This supports the promotion of universal access to genetic testing for Parkinson's disease, as well as therapeutic trials for GBA1 and LRRK2-related Parkinson's disease.
Assuntos
Testes Genéticos , Glucosilceramidase , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson , alfa-Sinucleína , Humanos , Doença de Parkinson/genética , Doença de Parkinson/diagnóstico , Testes Genéticos/métodos , Masculino , Feminino , Glucosilceramidase/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , alfa-Sinucleína/genética , Idoso , Pessoa de Meia-Idade , Ubiquitina-Proteína Ligases/genética , Proteínas Quinases/genética , Proteína Desglicase DJ-1/genética , Proteínas de Transporte Vesicular/genética , América do Norte , Variação Genética/genética , Predisposição Genética para Doença/genética , Adulto , Revelação , Aconselhamento Genético , Canadá , Estados UnidosRESUMO
BACKGROUND: Specific microglia responses are thought to contribute to the development and progression of neurodegenerative diseases, including Parkinson's disease (PD). However, the phenotypic acquisition of microglial cells and their role during the underlying neuroinflammatory processes remain largely elusive. Here, according to the multiple-hit hypothesis, which stipulates that PD etiology is determined by a combination of genetics and various environmental risk factors, we investigate microglial transcriptional programs and morphological adaptations under PARK7/DJ-1 deficiency, a genetic cause of PD, during lipopolysaccharide (LPS)-induced inflammation. METHODS: Using a combination of single-cell RNA-sequencing, bulk RNA-sequencing, multicolor flow cytometry and immunofluorescence analyses, we comprehensively compared microglial cell phenotypic characteristics in PARK7/DJ-1 knock-out (KO) with wildtype littermate mice following 6- or 24-h intraperitoneal injection with LPS. For translational perspectives, we conducted corresponding analyses in human PARK7/DJ-1 mutant induced pluripotent stem cell (iPSC)-derived microglia and murine bone marrow-derived macrophages (BMDMs). RESULTS: By excluding the contribution of other immune brain resident and peripheral cells, we show that microglia acutely isolated from PARK7/DJ-1 KO mice display a distinct phenotype, specially related to type II interferon and DNA damage response signaling, when compared with wildtype microglia, in response to LPS. We also detected discrete signatures in human PARK7/DJ-1 mutant iPSC-derived microglia and BMDMs from PARK7/DJ-1 KO mice. These specific transcriptional signatures were reflected at the morphological level, with microglia in LPS-treated PARK7/DJ-1 KO mice showing a less amoeboid cell shape compared to wildtype mice, both at 6 and 24 h after acute inflammation, as also observed in BMDMs. CONCLUSIONS: Taken together, our results show that, under inflammatory conditions, PARK7/DJ-1 deficiency skews microglia towards a distinct phenotype characterized by downregulation of genes involved in type II interferon signaling and a less prominent amoeboid morphology compared to wildtype microglia. These findings suggest that the underlying oxidative stress associated with the lack of PARK7/DJ-1 affects microglia neuroinflammatory responses, which may play a causative role in PD onset and progression.
Assuntos
Inflamação , Lipopolissacarídeos , Camundongos Knockout , Microglia , Proteína Desglicase DJ-1 , Animais , Proteína Desglicase DJ-1/deficiência , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/metabolismo , Microglia/metabolismo , Microglia/patologia , Microglia/efeitos dos fármacos , Camundongos , Lipopolissacarídeos/toxicidade , Lipopolissacarídeos/farmacologia , Inflamação/patologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/genética , Humanos , Camundongos Endogâmicos C57BL , Doenças Neuroinflamatórias/patologia , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/induzido quimicamente , Doenças Neuroinflamatórias/genéticaRESUMO
DJ-1, a causative gene for hereditary recessive Parkinsonism, is evolutionarily conserved across eukaryotes and prokaryotes. Structural analyses of DJ-1 and its homologs suggested the 106th Cys is a nucleophilic cysteine functioning as the catalytic center of hydratase or hydrolase activity. Indeed, DJ-1 and its homologs can convert highly electrophilic α-oxoaldehydes such as methylglyoxal into α-hydroxy acids as hydratase in vitro, and oxidation-dependent ester hydrolase (esterase) activity has also been reported for DJ-1. The mechanism underlying such plural activities, however, has not been fully characterized. To address this knowledge gap, we conducted a series of biochemical assays assessing the enzymatic activity of DJ-1 and its homologs. We found no evidence for esterase activity in any of the Escherichia coli DJ-1 homologs. Furthermore, contrary to previous reports, we found that oxidation inactivated rather than facilitated DJ-1 esterase activity. The E. coli DJ-1 homolog HchA possesses phenylglyoxalase and methylglyoxalase activities but lacks esterase activity. Since evolutionary trace analysis identified the 186th H as a candidate residue involved in functional differentiation between HchA and DJ-1, we focused on H186 of HchA and found that an esterase activity was acquired by H186A mutation. Introduction of reverse mutations into the equivalent position in DJ-1 (A107H) selectively eliminated its esterase activity without compromising α-oxoaldehyde hydratase activity. The obtained results suggest that differences in the amino acid sequences near the active site contributed to acquisition of esterase activity in vitro and provide an important clue to the origin and significance of DJ-1 esterase activity.
Assuntos
Escherichia coli , Doença de Parkinson , Proteína Desglicase DJ-1 , Proteína Desglicase DJ-1/metabolismo , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/química , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Esterases/metabolismo , Esterases/genética , Esterases/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Evolução Molecular , OxirreduçãoRESUMO
Acute kidney injury (AKI) is extremely prevalent among hospitalizations and presents a significant risk for the development of chronic kidney disease and increased mortality. Ischemia caused by shock, trauma, and transplant are common causes of AKI. To attenuate ischemic AKI therapeutically, we need a better understanding of the physiological and cellular mechanisms underlying damage. Instances of ischemia are most damaging in proximal tubule epithelial cells (PTECs) where hypoxic signaling cascades, and perhaps more rapidly, posttranslational modifications (PTMs), act in concert to change cellular metabolism. Here, we focus on the effects of the understudied PTM, lysine succinylation. We have previously shown a protective effect of protein hypersuccinylation on PTECs after depletion of the desuccinylase sirtuin5. General trends in the results suggested that hypersuccinylation led to upregulation of peroxisomal activity and was protective against kidney injury. Included in the list of changes was the Parkinson's-related deglycase Park7. There is little known about any links between peroxisome activity and Park7. In this study, we show in vitro and in vivo that Park7 has a crucial role in protection from AKI and upregulated peroxisome activity. These data in combination with published results of Park7's protective role in cardiovascular damage and chronic kidney disease lead us to hypothesize that succinylation of Park7 may ameliorate oxidative damage resulting from AKI and prevent disease progression. This novel mechanism provides a potential therapeutic mechanism that can be targeted.NEW & NOTEWORTHY Succinylation is an understudied posttranslational modification that has been shown to increase peroxisomal activity. Furthermore, increased peroxisomal activity has been shown to reduce oxidative stress and protect proximal tubules after acute kidney injury. Analysis of mass spectrometry succinylomic and proteomic data reveals a novel role for Parkinson's related Park7 in mediating Nrf2 antioxidant response after kidney injury. This novel protection pathway provides new insights for kidney injury prevention and development of novel therapeutics.
Assuntos
Injúria Renal Aguda , Túbulos Renais Proximais , Proteína Desglicase DJ-1 , Animais , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/prevenção & controle , Injúria Renal Aguda/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Proteína Desglicase DJ-1/metabolismo , Proteína Desglicase DJ-1/genética , Processamento de Proteína Pós-Traducional , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Masculino , Sirtuínas/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais , Camundongos , Estresse Oxidativo , Lisina/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) leads to death within 2-5 yr. Currently, available drugs only slightly prolong survival. We present novel insights into the pathophysiology of Superoxide Dismutase 1 (SOD1)- and in particular Fused In Sarcoma (FUS)-ALS by revealing a supposedly central role of glycolic acid (GA) and D-lactic acid (DL)-both putative products of the Parkinson's disease associated glyoxylase DJ-1. Combined, not single, treatment with GA/DL restored axonal organelle phenotypes of mitochondria and lysosomes in FUS- and SOD1-ALS patient-derived motoneurons (MNs). This was not only accompanied by restoration of mitochondrial membrane potential but even dependent on it. Despite presenting an axonal transport deficiency as well, TDP43 patient-derived MNs did not share mitochondrial depolarization and did not respond to GA/DL treatment. GA and DL also restored cytoplasmic mislocalization of FUS and FUS recruitment to DNA damage sites, recently reported being upstream of the mitochondrial phenotypes in FUS-ALS. Whereas these data point towards the necessity of individualized (gene-) specific therapy stratification, it also suggests common therapeutic targets across different neurodegenerative diseases characterized by mitochondrial depolarization.
Assuntos
Esclerose Lateral Amiotrófica , Glicolatos , Ácido Láctico , Mitocôndrias , Proteína Desglicase DJ-1 , Proteína FUS de Ligação a RNA , Superóxido Dismutase-1 , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/genética , Proteína FUS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/genética , Glicolatos/metabolismo , Glicolatos/farmacologia , Mitocôndrias/metabolismo , Proteína Desglicase DJ-1/metabolismo , Proteína Desglicase DJ-1/genética , Ácido Láctico/metabolismo , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase-1/genética , Potencial da Membrana Mitocondrial , Neurônios Motores/metabolismo , Lisossomos/metabolismoRESUMO
BACKGROUND: Ischemic postconditioning (IPostC) has been reported as a promising method for protecting against myocardial ischemia-reperfusion (MI/R) injury. Our previous study found that the infarct-limiting effect of IPostC is abolished in the heart of diabetes whose cardiac expression of DJ-1 (also called PARK7, Parkinsonism associated deglycase) is reduced. However, the role and in particular the underlying mechanism of DJ-1 in the loss of sensitivity to IPostC-induced cardioprotection in diabetic hearts remains unclear. METHODS: Streptozotocin-induced type 1 diabetic rats were subjected to MI/R injury by occluding the left anterior descending artery (LAD) and followed by reperfusion. IPostC was induced by three cycles of 10s of reperfusion and ischemia at the onset of reperfusion. AAV9-CMV-DJ-1, AAV9-CMV-C106S-DJ-1 or AAV9-DJ-1 siRNA were injected via tail vein to either over-express or knock-down DJ-1 three weeks before inducing MI/R. RESULTS: Diabetic rats subjected to MI/R exhibited larger infarct area, more severe oxidative injury concomitant with significantly reduced cardiac DJ-1 expression and increased PTEN expression as compared to non-diabetic rats. AAV9-mediated cardiac DJ-1 overexpression, but not the cardiac overexpression of DJ-1 mutant C106S, restored IPostC-induced cardioprotection and this effect was accompanied by increased cytoplasmic DJ-1 translocation toward nuclear and mitochondrial, reduced PTEN expression, and increased Nrf-2/HO-1 transcription. Our further study showed that AAV9-mediated targeted DJ-1 gene knockdown aggravated MI/R injury in diabetic hearts, and this exacerbation of MI/R injury was partially reversed by IPostC in the presence of PTEN inhibition or Nrf-2 activation. CONCLUSIONS: These findings suggest that DJ-1 preserves the cardioprotective effect of IPostC against MI/R injury in diabetic rats through nuclear and mitochondrial DJ-1 translocation and that inhibition of cardiac PTEN and activation of Nrf-2/HO-1 may represent the major downstream mechanisms whereby DJ-1 preserves the cardioprotective effect of IPostC in diabetes.
Assuntos
Diabetes Mellitus Experimental , Pós-Condicionamento Isquêmico , Traumatismo por Reperfusão Miocárdica , PTEN Fosfo-Hidrolase , Proteína Desglicase DJ-1 , Ratos Sprague-Dawley , Animais , Proteína Desglicase DJ-1/metabolismo , Proteína Desglicase DJ-1/genética , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Diabetes Mellitus Experimental/metabolismo , Masculino , Ratos , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/complicações , Transporte Proteico , Estreptozocina , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologiaRESUMO
Diallyl disulfide (DADS), an organic component of allicin abstracted from garlic, possesses multi-target antitumor activity. DJ-1 performs a vital function in promoting AKT aberrant activation via down-regulating phosphatase and tensin homologue (PTEN) in tumors. It is unknown the involvement of DJ-1 in epithelial-mesenchymal transition (EMT) of gastric cancer (GC) cells. The purpose of this study is to investigate whether diallyl disulfide (DADS) intervenes in the role of DJ-1 in GC. Based on the identification that the correlation between high DJ-1 and low PTEN expression in GC was implicated in clinical progression, we illuminated that down-regulation of DJ-1 by DADS aided in an increase in PTEN expression and a decrease in phosphorylated AKT levels, which was in line with the results manifested in the DJ-1 knockdown and overexpressed cells, concurrently inhibiting proliferation, EMT, migration, and invasion. Furthermore, the antagonistic effects of DADS on DJ-1 were observed in in vivo experiments. Additionally, DADS mitigated the DJ-1-associated drug resistance. The current study revealed that DJ-1 is one of potential targets for DADS, which hopefully provides a promising strategy for prevention and adjuvant chemotherapy of GC.
Assuntos
Compostos Alílicos , Proliferação de Células , Dissulfetos , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Proteína Desglicase DJ-1 , Neoplasias Gástricas , Dissulfetos/farmacologia , Proteína Desglicase DJ-1/metabolismo , Proteína Desglicase DJ-1/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Compostos Alílicos/farmacologia , Humanos , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Animais , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Movimento Celular/efeitos dos fármacos , Camundongos , Camundongos Nus , Camundongos Endogâmicos BALB CRESUMO
Regulation of the oxidative stress response is crucial for the management and prognosis of traumatic brain injury (TBI). The copper chaperone Antioxidant 1 (Atox1) plays a crucial role in regulating intracellular copper ion balance and impacting the antioxidant capacity of mitochondria, as well as the oxidative stress state of cells. However, it remains unknown whether Atox1 is involved in modulating oxidative stress following TBI. Here, we investigated the regulatory role of Atox1 in oxidative stress on neurons both in vivo and in vitro, and elucidated the underlying mechanism through culturing hippocampal HT-22 cells with Atox1 mutation. The expression of Atox1 was significantly diminished following TBI, while mice with overexpressed Atox1 exhibited a more preserved hippocampal structure and reduced levels of oxidative stress post-TBI. Furthermore, the mice displayed notable impairments in learning and memory functions after TBI, which were ameliorated by the overexpression of Atox1. In the stretch injury model of HT-22 cells, overexpression of Atox1 mitigated oxidative stress by preserving the normal morphology and network connectivity of mitochondria, as well as facilitating the elimination of damaged mitochondria. Mechanistically, co-immunoprecipitation and mass spectrometry revealed the binding of Atox1 to DJ-1. Knockdown of DJ-1 in HT-22 cells significantly impaired the antioxidant capacity of Atox1. Mutations in the copper-binding motif or sequestration of free copper led to a substantial decrease in the interaction between Atox1 and DJ-1, with overexpression of DJ-1 failing to restore the antioxidant capacity of Atox1 mutants. The findings suggest that DJ-1 mediates the ability of Atox1 to withstand oxidative stress. And targeting Atox1 could be a potential therapeutic approach for addressing post-traumatic neurological dysfunction.
Assuntos
Lesões Encefálicas Traumáticas , Proteínas de Transporte de Cobre , Hipocampo , Mitofagia , Neurônios , Estresse Oxidativo , Proteína Desglicase DJ-1 , Animais , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Lesões Encefálicas Traumáticas/genética , Camundongos , Hipocampo/metabolismo , Hipocampo/patologia , Neurônios/metabolismo , Proteína Desglicase DJ-1/metabolismo , Proteína Desglicase DJ-1/genética , Proteínas de Transporte de Cobre/metabolismo , Proteínas de Transporte de Cobre/genética , Mitocôndrias/metabolismo , Modelos Animais de Doenças , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Masculino , Antioxidantes/metabolismo , Linhagem Celular , HumanosRESUMO
Genome editing with CRISPR RNA-guided endonucleases generates DNA breaks that are resolved by cellular DNA repair machinery. However, analogous methods to manipulate RNA remain unavailable. We show that site-specific RNA breaks generated with type-III CRISPR complexes are repaired in human cells and that this repair can be used for programmable deletions in human transcripts to restore gene function. Collectively, this work establishes a technology for precise RNA manipulation with potential therapeutic applications.
Assuntos
Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Edição de Genes , RNA Guia de Sistemas CRISPR-Cas , RNA , Humanos , Reparo do DNA , Endonucleases/metabolismo , Edição de Genes/métodos , Células HEK293 , RNA/genética , RNA Guia de Sistemas CRISPR-Cas/genética , Proteína Desglicase DJ-1/genética , Ciclofilinas/genética , Streptococcus thermophilusRESUMO
Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are the world's leading causes of blindness. The retinal pigment epithelium (RPE) and vascular endothelial cell exposed to oxidative stress is the major cause of AMD and DR. DJ-1, an important endogenous antioxidant, its overexpression is considered as a promising antioxidant treatment for AMD and DR. Here, we modified the tetrahedral frame nucleic acids (tFNAs) with DJ-1 saRNAs as a delivery system, and synthesized a novel nanocomplex (tFNAs-DJ-1 saRNAs). In vitro studies show that tFNAs-DJ-1 saRNAs can efficiently transfer DJ-1 saRNAs to human umbilical vein endothelial cells (HUVECs) and ARPE-19s, and significantly increased their cellular DJ-1 level. Reactive oxygen species expression in H2O2-treated HUVECs and ARPE-19s were decreased, cell viability was enhanced and cell apoptosis were inhibited when tFNAs-DJ-1 saRNAs were delivered. Moreover, tFNAs-DJ-1 saRNAs preserved mitochondrial structure and function under oxidative stress conditions. In the aspect of molecular mechanism, tFNAs-DJ-1 saRNAs activated Erk and Nrf2 pathway, which might contribute to its protective effects against oxidative stress damage. To conclude, this study shows the successfully establishment of a simple but effective delivery system of DJ-1 saRNAs associated with antioxidant effects in AMD and DR, which may be a promising agent for future treatment in oxidative stress-related retinal disorders.
Assuntos
Antioxidantes , Células Endoteliais da Veia Umbilical Humana , Estresse Oxidativo , Proteína Desglicase DJ-1 , Humanos , Estresse Oxidativo/efeitos dos fármacos , Proteína Desglicase DJ-1/metabolismo , Proteína Desglicase DJ-1/genética , Antioxidantes/farmacologia , Antioxidantes/química , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Retina/metabolismo , Retina/efeitos dos fármacos , Ácidos Nucleicos/farmacologia , Ácidos Nucleicos/metabolismo , Linhagem CelularRESUMO
BACKGROUND: GBA1 mutations are the most common genetic risk factor for development of Parkinson's disease (PD). The loss of catalytic activity in GBA1, as well as the reduction of the GBA1 protein in certain cellular compartment, may increase disease progression. However, the mechanisms underlying cellular dysfunction caused by GBA1 deficiency are still mostly unknown. OBJECTIVE: In this study, we focus on the genetic interaction between GBA1 deficiency and PD-causing genes, such as DJ-1, in mitochondrial dysfunction. METHODS: GBA1 knockout (KO) SH-SY5Y cells were used to assess DJ-1 functions against oxidative stress in vitro. The levels of cellular reactive oxygen species were monitored with MitoSOX reagent. The expression of the PARK7 gene was analyzed using the quantitative real-time PCR (qRT-PCR). To understand the mechanism underlying DJ-1 upregulation in GBA1 KO cells, we assess ROS levels, antioxidant protein, and cell viability in GBA1 KO cells with treatment of ROS inhibitor N-acetyl-cysteine or miglustat, which is an inhibitor of glucosylceramide synthase. Dopaminergic degeneration was assessed from Gba1 L444P heterozygous mice mated with Park7 knockout mice. RESULTS: We find that DJ-1 is significantly upregulated in GBA1 KO cells. Elevated levels of DJ-1 are attributed to the transcriptional expression of PARK7 mRNA, but not the inhibition of DJ-1 protein degradation. Because DJ-1 expression is highly linked to oxidative stress, we observe cellular reactive oxygen species (ROS) in GBA1 KO cells. Moreover, several antioxidant gene expressions and protein levels are increased in GBA1 KO cells. To this end, GBA1 KO cells are more susceptible to H2O2-induced cell death. Importantly, there is a significant reduction in dopaminergic neurons in the midbrain from Gba1 L444P heterozygous mice mated with Park7 knockout mice, followed by mild motor dysfunction. CONCLUSION: Taken together, our results suggest that DJ-1 upregulation due to GBA1 deficiency has a protective role against oxidative stress. It may be supposed that mutations or malfunctions in the DJ-1 protein may have disadvantages in the survival of dopaminergic neurons in the brains of patients harboring GBA1 mutations.
Assuntos
Antioxidantes , Neuroblastoma , Doença de Parkinson , Humanos , Camundongos , Animais , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/metabolismo , Peróxido de Hidrogênio , Estresse Oxidativo , Morte Celular/fisiologia , Camundongos Knockout , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/metabolismoRESUMO
DJ-1, also known as Parkinson's disease protein 7 (Park7), is a multifunctional protein that regulates oxidative stress and mitochondrial function. Dysfunction of DJ-1 is implicated in the pathogenesis of Parkinson's disease (PD). Hyperhomocysteinemia is associated with an increased risk of PD. Here we show that homocysteine thiolactone (HTL), a reactive thioester of homocysteine (Hcy), covalently modifies DJ-1 on the lysine 182 (K182) residue in an age-dependent manner. The N-homocysteinylation (N-hcy) of DJ-1 abolishes its neuroprotective effect against oxidative stress and mitochondrial dysfunction, exacerbating cell toxicity. Blocking the N-hcy of DJ-1 restores its protective effect. These results indicate that the N-hcy of DJ-1 abolishes its neuroprotective effect and promotes the progression of PD. Inhibiting the N-hcy of DJ-1 may exert neuroprotective effect against PD.
Assuntos
Homocisteína , Doença de Parkinson , Proteína Desglicase DJ-1 , Proteína Desglicase DJ-1/metabolismo , Proteína Desglicase DJ-1/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Homocisteína/metabolismo , Homocisteína/análogos & derivados , Humanos , Animais , Estresse Oxidativo/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismoRESUMO
BACKGROUND: Liver fibrosis can progress to cirrhosis and hepatic carcinoma without treatment. CircDCBLD2 was found to be downregulated in liver fibrosis. However, the precise underlying mechanism requires further investigation. METHODS: qRT-PCR, Western blot, and immunohistochemistry assays were used to detect the related molecule levels. HE, Masson's trichrome, and Sirius Red staining were used to assess the pathological changes in mice's liver tissues. Flow cytometric analysis and commercial kit were used to assess the levels of lipid reactive oxygen species (ROS), malonaldehyde (MDA), glutathione (GSH), and iron. Cell viability was assessed by MTT. Immunoprecipitation was used to study the ubiquitination of PARK7. Mitophagy was determined by immunostaining and confocal imaging. RIP and Co-IP assays were used to assess the interactions of circDCBLD2/HuR, HuR/STUB1, and STUB1/PARK7. Fluorescence in situ hybridization and immunofluorescence staining were used to assess the co-localization of circDCBLD2 and HuR. RESULTS: CircDCBLD2 was downregulated, whereas PARK7 was upregulated in liver fibrosis. Ferroptosis activators increased circDCBLD2 while decreasing PARK7 in hepatic stellate cells (HSCs) and mice with liver fibrosis. CircDCBLD2 overexpression reduced cell viability and GSH, PARK7, and GPX4 expression in erastin-treated HSCs while increasing MDA and iron levels, whereas circDCBLD2 knockdown had the opposite effect. CircDCBLD2 overexpression increased STUB1-mediated PARK7 ubiquitination by promoting HuR-STUB1 binding and thus increasing STUB1 mRNA stability. PARK7 overexpression or HuR knockdown reversed the effects of circDCBLD2 overexpression on HSC activation and ferroptosis. CircDCBLD2 reduced liver fibrosis in mice by inhibiting PARK7. CONCLUSION: CircDCBLD2 overexpression increased PARK7 ubiquitination degradation by upregulating STUB1 through its interaction with HuR, inhibiting HSC activation and promoting HSC ferroptosis, ultimately enhancing liver fibrosis.
Assuntos
Ferroptose , Neoplasias Hepáticas , Animais , Camundongos , Células Estreladas do Fígado/metabolismo , Hibridização in Situ Fluorescente , Ferro/metabolismo , Ferro/farmacologia , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/metabolismo , Proteína Desglicase DJ-1/farmacologia , UbiquitinaçãoRESUMO
Parkinson's disease (PD) is a common movement disorder associated with the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Mutations in the PD-associated gene PARK7 alter the structure and function of the encoded protein DJ-1, and the resulting autosomal recessively inherited disease increases the risk of developing PD. DJ-1 was first discovered in 1997 as an oncogene and was associated with early-onset PD in 2003. Mutations in DJ-1 account for approximately 1% of all recessively inherited early-onset PD occurrences, and the functions of the protein have been studied extensively. In healthy subjects, DJ-1 acts as an antioxidant and oxidative stress sensor in several neuroprotective mechanisms. It is also involved in mitochondrial homeostasis, regulation of apoptosis, chaperone-mediated autophagy (CMA), and dopamine homeostasis by regulating various signaling pathways, transcription factors, and molecular chaperone functions. While DJ-1 protects neurons against damaging reactive oxygen species, neurotoxins, and mutant α-synuclein, mutations in the protein may lead to inefficient neuroprotection and the progression of PD. As current therapies treat only the symptoms of PD, the development of therapies that directly inhibit oxidative stress-induced neuronal cell death is critical. DJ-1 has been proposed as a potential therapeutic target, while oxidized DJ-1 could operate as a biomarker for PD. In this paper, we review the role of DJ-1 in the pathogenesis of PD by highlighting some of its key neuroprotective functions and the consequences of its dysfunction.
Assuntos
Doença de Parkinson , Humanos , Doença de Parkinson/metabolismo , Estresse Oxidativo/genética , Antioxidantes/metabolismo , Neurônios Dopaminérgicos/metabolismo , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/metabolismoRESUMO
BACKGROUND: Melanoma, the most lethal skin cancer type, occurs more frequently in Parkinson's disease (PD), and PD is more frequent in melanoma patients, suggesting disease mechanisms overlap. α-synuclein, a protein that accumulates in PD brain, and the oncogene DJ-1, which is associated with PD autosomal recessive forms, are both elevated in melanoma cells. Whether this indicates melanoma progression or constitutes a protective response remains unclear. We hereby investigated the molecular mechanisms through which α-synuclein and DJ-1 interact, suggesting novel biomarkers and targets in melanoma. METHODS: The Cancer Genome Atlas (TCGA) expression profiles derived from UCSC Xena were used to obtain α-synuclein and DJ-1 expression and correlated with survival in skin cutaneous melanoma (SKCM). Immunohistochemistry determined the expression in metastatic melanoma lymph nodes. Protein-protein interactions (PPIs) and molecular docking assessed protein binding and affinity with chemotherapeutic drugs. Further validation was performed using in vitro cellular models and ELISA immunoassays. RESULTS: α-synuclein and DJ-1 were upregulated in primary and metastatic SKCM. Aggregated α-synuclein was selectively detected in metastatic melanoma lymph nodes. α-synuclein overexpression in SK-MEL-28 cells induced the expression of DJ-1, supporting PPI and a positive correlation in melanoma patients. Molecular docking revealed a stable protein complex, with differential binding to chemotherapy drugs such as temozolomide, dacarbazine, and doxorubicin. Parallel reduction of both proteins in temozolomide-treated SK-MEL-28 spheroids suggests drug binding may affect protein interaction and/or stability. CONCLUSION: α-synuclein, together with DJ-1, may play a role in melanoma progression and chemosensitivity, constituting novel targets for therapeutic intervention, and possible biomarkers for melanoma.
Assuntos
Biomarcadores Tumorais , Melanoma , Proteína Desglicase DJ-1 , Neoplasias Cutâneas , alfa-Sinucleína , Humanos , Proteína Desglicase DJ-1/metabolismo , Proteína Desglicase DJ-1/genética , Melanoma/metabolismo , Melanoma/patologia , Melanoma/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/genética , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Linhagem Celular Tumoral , Biomarcadores Tumorais/metabolismo , Simulação de Acoplamento Molecular , Regulação Neoplásica da Expressão Gênica , Masculino , Metástase Linfática , Ligação Proteica , FemininoRESUMO
Diffuse large B-cell lymphoma (DLBCL) is difficult to treat due to the high recurrence rate and therapy intolerance, so finding potential therapeutic targets for DLBCL is critical. FK506-binding protein 3 (FKBP3) contributes to the progression of various cancers and is highly expressed in DLBCL, but the role of FKBP3 in DLBCL and its mechanism are not clear. Our study demonstrated that FKBP3 aggravated the proliferation and stemness of DLBCL cells, and tumour growth in a xenograft mouse model. The interaction between FKBP3 and parkinsonism associated deglycase (PARK7) in DB cells was found using co-immunoprecipitation assay. Knockdown of FKBP3 enhanced the degradation of PARK7 through increasing its ubiquitination modification. Forkhead Box O3 (FOXO3) belongs to the forkhead family of transcription factors and inhibits DLBCL, but the underlying mechanism has not been reported. We found that FOXO3 bound the promoter of FKBP3 and then suppressed its transcription, eventually weakening DLBCL. Mechanically, FKBP3 activated Wnt/ß-catenin signalling pathway mediated by PARK7. Together, FKBP3 increased PARK7 and then facilitated the malignant phenotype of DLBCL through activating Wnt/ß-catenin pathway. These results indicated that FKBP3 might be a potential therapeutic target for the treatment of DLBCL.
Assuntos
Linfoma Difuso de Grandes Células B , beta Catenina , Humanos , Camundongos , Animais , beta Catenina/metabolismo , Proteína Desglicase DJ-1/genética , Regulação Neoplásica da Expressão Gênica , Via de Sinalização Wnt/genética , Fenótipo , Linfoma Difuso de Grandes Células B/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Association between cancer risk and Parkinson's disease is still debated. DJ-1, a Parkinson's disease (PD)-related gene, is encoded by PARK-7 gene and its deficiency causes early-onset PD. In our last studies, it was found that the immunosuppressive microenvironment established in DJ-1 knockout (KO) mice can enhance metastasis of melanoma cells to lungs. Therefore, we wanted to further examine whether there were some niche in other organs of DJ-1-deficiency mouse to facilitate cell growth of tumors. We used in vivo tissue-specific models of tumor growth and in vitro cellular model to verify the hypothesis. We also used protein blot assay, cell-adhesion assay and bioinformatic tools to conduct experiments. In the mouse model of subcutaneous injection, there was no difference on tumor growth between WT and DJ-1 KO mice. Moreover, the results of experimental liver metastasis by intrasplenic injection model showed that there was no difference of nodules number in both mice, but a dramatic enhancement of nodule formation and increased mucin4 levels were found in pancreas of DJ-1 KO mice. In cell cultures, we further found that B16F10 cells indeed tended to adhere well to primary DJ-1-deficiency pancreatic epithelial cells, which had higher protein levels of mucin4. Notably, a human database also showed the inverse relationship in human pancreas between DJ-1 and mucin4, and mucin4 down-regulation can reverse the enhanced cellular adhesion in DJ-1 KO pancreatic epithelial cells. These results indicated that DJ-1 KO pancreatic tissue creating an appropriate microenvironment benefited development of the cancer cells.
Assuntos
Neoplasias , Doença de Parkinson , Animais , Humanos , Camundongos , Pulmão/metabolismo , Camundongos Knockout , Pâncreas/metabolismo , Doença de Parkinson/metabolismo , Proteína Desglicase DJ-1/genética , Microambiente TumoralRESUMO
Parkinson's disease (PD) is an incurable neurodegenerative disorder caused by the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Current therapies are only symptomatic and are not able to stop or delay its progression. In order to search for new and more effective therapies, our group carried out a high-throughput screening assay, identifying several candidate compounds that are able to improve locomotor ability in DJ-1ß mutant flies (a Drosophila model of familial PD) and reduce oxidative stress (OS)-induced lethality in DJ-1-deficient SH-SY5Y human cells. One of them was vincamine (VIN), a natural alkaloid obtained from the leaves of Vinca minor. Our results showed that VIN is able to suppress PD-related phenotypes in both Drosophila and human cell PD models. Specifically, VIN reduced OS levels in PD model flies. Besides, VIN diminished OS-induced lethality by decreasing apoptosis, increased mitochondrial viability, and reduced OS levels in DJ-1-deficient human cells. In addition, our results show that VIN might be exerting its beneficial role, at least partially, by the inhibition of voltage-gated sodium channels. Therefore, we propose that these channels might be a promising target in the search for new compounds to treat PD and that VIN represents a potential therapeutic treatment for the disease.